Biology subjects

Flechsig, H.

Publications and source records attributed to Flechsig, H..

15 recordsLinked to original sources

bioRxiv2026

Dual role of the RIG-I 2CARD as a signaling module and a brake on dsRNA translocation

RIG-I is an innate immune receptor for viral double-stranded RNA (dsRNA). Upon binding dsRNA, RIG-I nucleates MAVS filaments through its two caspase activation and recruitment domains (2CARD). However, how the 2CARD behaves during activation has remained unclear. Using high-speed atomic force microscopy, we visualized the 2CARD in full-length RIG-I across its apo, dsRNA-bound, translocating, multimeric, and polyubiquitin-bound states. Protrusions consistent with the 2CARD appeared only transiently in the apo state, whereas a mobile 2CARD emerged upon RNA binding. During ATP-driven translocation, wild-type RIG-I moved approximately twofold slower and showed more frequent pauses and multimers than RIG-I {Delta}2CARD. Within multimers, 2CARDs formed a complex that polyubiquitin stabilized into larger, dsRNA-deforming assemblies. These findings reveal the 2CARD as a brake on the RIG-I motor, beyond signaling.

biophysics↗

bioRxiv2026

BioAFMviewer AFM Analysis Arena A3 - an interactive software platform for automated analysis of HS-AFM imaging

High-speed atomic force microscopy (HS-AFM) experiments provide unique insights into nanoscale biological processes by directly visualizing the surface topography dynamics of biomolecules, such as proteins, over extended periods of time and generating large imaging data sets. However, the lack of computational methods for automated data analysis has thus far limited the full utilization of these data for the quantitative understanding of single-molecule dynamic. Here, we present the AFM Analysis Arena (A3) within BioAFMviewer as a software platform for automated, high-throughput quantitative analysis of AFM topographic imaging data within a user-friendly interactive graphical interface, implementing a convenient workflow from raw HS-AFM (AFM) imaging to statistical analysis. In several applications to HS-AFM imaging data, we demonstrate methods of automatized molecule detection and dynamic tracking to highlight the available capacities, aiming to transform HS-AFM measurements from observations towards an analytical tool in nanoscale biology.

biophysics↗

bioRxiv2026

Coiled-coil domain kinking controls laminin-332 cleavage by elastase

Laminins are trimeric glycoproteins and essential components of basement membranes, where they provide structural support and mediate cell-matrix adhesion. Their -, {beta}-, and {gamma}-chains combine into a flexible trimeric coiled-coil domain forming the laminin long arm, which links the N-terminal short arms and the C-terminal globular domains. However, ultrastructural insight into the laminin coiled-coil domain remains limited, and the function of coiled-coil flexibility remains unclear. Using high-speed atomic force microscopy (HS-AFM) imaging, we previously investigated the ultrastructure of laminin-332 and observed dynamic kinking around a putative molecular hinge in the center of the coiled-coil domain. In this study, we integrate HS-AFM time-lapse imaging with AlphaFold structure prediction and normal-mode flexible fitting (NMFF) to generate dynamic models of the laminin-332 hinge during coiled-coil domain kinking, providing atomic-scale insight into the underlying molecular rearrangements. Furthermore, we use HS-AFM to visualize, in real time, the digestion of individual laminin-332 molecules by pancreatic elastase and show that coiled-coil kinking directs elastase cleavage to the hinge site, thereby ensuring reliable generation of the elastase 8 (E8) fragment containing the integrin binding site. In contrast, laminin molecules with extended coiled-coil conformations are cleaved at arbitrary sites, resulting in complete coiled-coil removal and degradation. Our results thus identify a novel conformational control mechanism directing proteolytic processing of laminin-332 through defined coiled-coil kinking. Besides expanding the understanding of dynamic laminin coiled-coil function, the gained ultrastructural insight into trimeric coiled-coil bending may aid the rational design of flexible coiled-coil modules for future protein engineering applications.

cell biology↗

bioRxiv2025

Energy conversion mechanism revealed by ATP-free and ATP-1 dependent walking of myosin V motor

The mechanical processes of ATPase protein motors are coupled to their ATPase reactions. The free energy released at a given step(s) of the ATPase cycle is converted into mechanical work. Therefore, it has long been thought that ATP is the energy source for mechanical work. Here, we show that the double-headed myosin V (M5) motor can perform ATP-free walking. In the presence of ADP, M5 bound to F-actin by both heads makes a forward step when the trailing head is detached by an external force. This forward step harnesses the strain energy in the leading (L) head, provided from the strong binding energy between the L-head and actin. Consequently, strain generation reduces the binding affinity, which manifests as sporadic, brief L-head detachment. Strain generation and brief L-head detachment also occur during ATP-dependent walking to similar extents as during ATP-free walking. These results suggest that the strong binding energy is also converted into mechanical work during ATP-dependent walking.

biophysics↗

bioRxiv2025

High-Speed AFM Reveals IDR-Mediated Structural Plasticity of EML4-ALK Modulated by ALK Inhibitors

EML4-ALK is a key oncogenic driver in lung cancer, but variant-specific chemoresistance limits the efficacy of current ALK inhibitors. Because the N-terminus contains an intrinsically disordered region (IDR), how ALK inhibitors affect the structure and dynamics of full-length EML4-ALK remains unclear. Here, using high-speed atomic force microscopy (HS-AFM), we visualize the overall structures of three full-length EML4-ALK variants (v1, v3, and v5) at the single-molecule level. We identified a transient globular subdomain (residues 191-217) within the v3 IDR that contributes to distinct oligomerization patterns. Notably, ALK inhibitors compact the IDR subdomain and reduce oligomerization, whereas this effect is abolished by the resistance mutation ALKG1202R, suggesting that ALK inhibitors not only affect kinase activity but also modulate IDR dynamics. Our findings shed new light on the structural regulation of EML4-ALK and provide a foundation for developing next-generation targeted therapies that account for IDR-driven molecular behavior. TeaserHigh-speed AFM revealed the full-length EML4-ALK structures and their IDR-mediated plasticity caused by ALK kinase inhibitors

cancer biology↗

bioRxiv2025

Flexible fitting to infer atomistic-precision models of large-amplitude conformational dynamics in biomolecules from high-speed atomic force microscopy imaging

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes of the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton size protein filament demonstrate versatility of NMFF-AFM modelling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step towards automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the standalone user-friendly interactive BioAFMviewer software opens the opportunity for broad range applications to facilitate the understanding of resolution-limited HS-AFM measurements.

biophysics↗

bioRxiv2025

Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of CaMKII and CaMKII{beta} variants in the brain. Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKII/{beta} heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKII/{beta} at a 3:1 ratio. Our findings revealed that the CaMKII and CaMKII{beta} subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKII{beta} subunits adjacent to one another. Furthermore, in the activated state, CaMKII/{beta} heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKII{beta} subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKII{beta} subunits within the CaMKII/{beta} heterododecamer.

neuroscience↗

bioRxiv2023

High-speed AFM reveals fluctuations and dimer splitting of the N-terminal domain of GluA2-γ2

AMPA glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which participate in AMPAR clustering at synapses. Nevertheless, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit with TARP {gamma}2 in lipid environments. HS-AFM videos of GluA2-{gamma}2 in the resting and open states revealed fluctuations in NTD dimers. Conversely, in the desensitized state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we visualized individual NTD dimers transitioning into monomers. Furthermore, this NTD-dimer splitting resulted in intersubunit exchange between NTD dimers and an increased number of binding sites with the synaptic protein neuronal pentraxin 1. Therefore, our findings illuminate the significance of NTD dynamics in the synaptic clustering of AMPARs.

neuroscience↗

bioRxiv2023

Atom filtering algorithm and GPU-accelerated calculation of simulation atomic force microscopy images

Simulation atomic force microscopy computationally emulates experimental scanning of a biomolecular structure to produce topographic images that can be correlated with measured images. Its application to the enormous amount of available high-resolution structures, as well as to molecular dynamics modelling data, facilitates the quantitative interpretation of experimental observations by inferring atomistic information from resolution-limited measured topographies. The computation required to generate a simulated AFM image generally includes the calculation of contacts between the scanning tip and all atoms from the biomolecular structure. However, since only contacts with surface atoms are relevant, a filtering method shall highly improve the efficiency of simulation AFM computations. In this report we address this issue and present an elegant solution based on graphics processing unit (GPU) computations that significantly accelerates the computation of simulation AFM images. The method not only allows for visualization of biomolecular structures combined with ultra-fast synchronized calculation and graphical representation of corresponding simulated AFM images (live simulation AFM), but, as we demonstrate, can also reduce the computational effort during automatized fitting of atomistic structures into measured AFM topographies by orders of magnitude. Hence, the developed method will play an important role in post-experimental computational analysis involving simulation AFM. Implementation is realized in our BioAFMviewer software package for simulation AFM of biomolecular structures and dynamics.

biophysics↗

bioRxiv2023

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, and the ClpB molecular chaperone. The model also allows predictions of imaging stability under tip-scanning. We implemented the developed method within the freely available BioAFMviewer software package. Therefore, predictions based on available structural data can be made even prior to an actual experiment.

biophysics↗

bioRxiv2023

Evolutionarily acquired activity-dependent transformation of the CaMKII holoenzyme

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been central in synaptic plasticity research. CaMKII is a dodecameric serine/threonine kinase that has been essentially conserved across metazoans for over a million years. While the mechanisms of CaMKII activation are well studied, its "behavior" at the molecular level has remained unobserved. Here, high-speed atomic force microscopy was used to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII in various states at nanometer resolution. Among the species, rat CaMKII underwent internal kinase domain aggregation in an activity-dependent manner and showed a higher tolerance to dephosphorylation by phosphatase. Our findings suggest that mammalian CaMKII has evolutionarily acquired a new structural form and a tolerance to phosphatase to maintain robust CaMKII activity for proper neuronal function. One-Sentence SummaryHigh-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII

neuroscience↗

bioRxiv2022

Structural dynamics of E6AP E3 ligase HECT domain and involvement of flexible hinge loop in ubiquitin chain synthesis mechanism

Ubiquitin (Ub) ligases E3 are an important factor in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe containing the E2-binding site and a C-lobe containing the catalytic Cys residue that forms a thioester bond with Ub. These two lobes are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of this type of E3 ligases remain unclear. Here, we provide the first direct demonstration of structural dynamics of the E6AP HECT domain using high-speed atomic force microscopy. We also investigated structural dynamics of hinge loop flexibility restricted HECT domain, and we found that flexibility of the E6AP hinge loop has a great impact not only on its structural dynamics but also on the formation of free Ub chains mediated by E3-E3 interactions.

biophysics↗

bioRxiv2021

Simulation atomic force microscopy to predict correlated conformational dynamics in proteins from topographic imaging

Atomic force microscopy (AFM) of proteins can detect only changes within the scanned molecular surface, missing all motions in other regions and thus information about functionally relevant conformational couplings. We show that simulation AFM can overcome this drawback by reconstruction of 3D molecular structures from topographic AFM images. A proof of principle demonstration is provided for an in-silico AFM experiment visualizing the conformational dynamics of a membrane transporter. The application shows that the alternating access mechanism underlying its operation can be retrieved from only AFM imaging of one membrane side. Simulation AFM is implemented in the freely available BioAFMviewer software platform, providing the convenient applicability to better understand experimental AFM observations.

biophysics↗

bioRxiv2021

Atomic reconstruction of biomolecular structures from AFM images and quantitative validation of experimental data using simulated AFM scanning

We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.

biophysics↗

bioRxiv2019

Biomolecular AFM viewer

We developed a stand-alone software which allows to transform biomolecular structures and movies of their conformational dynamics into a representation which corresponds to the outcome of biomolecular atomic force microscopy (AFM) experiments, such as high-speed AFM. The software implements a high degree of usability. An integrated highly versatile molecular viewer allows the visualization of structures and their corresponding simulated AFM representations in arbitrary orientations. The obtained results can be conveniently exported as still images and movies. We provide a demonstration of our biomolecular AFM viewer by applying it to several proteins from the Protein Data Bank, and to a molecular movie of conformational transitions between two protein structures obtained from a modelling server.

biophysics↗